ABSTRACT
BACKGROUND: Development of liver fibrosis accompanies many morphological and functional changes. The pathological alterations of dimethylnitrosamine (DMN)-induced liver fibrosis in rats are similar to those of human liver fibrosis.OBJECTIVE: To observe the dynamic changes in morphology and serum hyaluronic acid (HA), laminin (LN), and type Ⅳ collagen in rats with liver fibrosis induced by DMN.DESIGN: Randomized controlled animal trial.SETTING: Laboratory of Teaching and Research Section of Pathology, College of Medicine, Yanbian University.MATERIALS: Eighty 3-month-old male rats of clean grade with 175-200 g body mass were selected, which were provided by the animal center of College of Medicine, Yanbian University. Agents: Dimethylnitrosamine provided by Sigma company, α smooth muscle actin by Dako company, Sirius red by Aldrich chem company, serum hyaluronic acid,laminin and type Ⅳ collagen kit by Sino-American Biotechnology Company, rabbit-anti-rat I g by Dako, Denmak company. Devices: JEM-1200EX transmission electron microscope made in Japan; enzyme linked immuno analyzer made in Japan; and CMTAS multifunction true color pathological image analysis system developed by Beijing University of Aeronautics and Astronautics.METHODS: The experiment was conducted in the College of Medicine, Yanbian University from June 2004 to December 2005. The rats were divided into 2 groups by lot: model group (n =40): The rats were intraperioneally injected with 10 g/L DMN (10 μL/kg) once daily, 3 days a week for 4 weeks; control group (n =40): The matching normal saline was injected at the same period; the blood from the left ventricle was collected and frozen in refrigerator at -70 ℃ before the rats were killed at days 7, 14, 21, and 28 and the liver tissue was removed for electron and light microscope observation. ①The dynamic changes in the content of serum HA, LN and type Ⅳ collagen were measured by enzyme-linked immunoabsorbent assay (ELISA). ②The morphological changes and liver fibrosis grading were examined by HE staining,immunohistochemical Sirius-red staining (liver fibrosis degree was classified into 5 grades: grade 0: no fibrosis; grade 1:fibrosis in portal area; grade 2: fibrotic septa between portal tracts; grade 3: fibrosis septa and structure disturbance of hepatic lobule; grade 4: cirrhosis), meanwhile, the area-density percentage of collagen fibrosis was calculated. ③The hepatic stellate cells were detected with transmission electron microscope and immunohistochemical alpha smooth muscle actin (SMA) staining. ④The correlation between area-density percentage of collagen fibrosis during liver fibrosis formation and the serum levels of HA, LN and type Ⅳ collagen was analyzed.MAIN OUTCOME MEASURES: ①The changes in the serum levels of HA, type Ⅳ collagen and LN during liver fibrosis formation; ②The morphological changes and liver fibrosis grading and area-density percentage of collagen fibrosis; ③Transformation and distribution characteristics of hepatic stellate cells; ④The correlation between area-density percentage of collagen fibrosis and the serum levels of HA, LN and type Ⅳ collagen.RESULTS: Among the 80 rats, 34 of the experimental group were modeled successfully, which were involved in the result analysis with the 40 rats in the control group. ①The levels of serum HA, type Ⅳ collagen and LN of the model group were significantly higher compared with the control group from day 7 to 28 (P ＜ 0.05), especially that on the 28th day. ②In the model group, the portal area of the rats showed hemorrhagic necrosis at day 7 after injection of DMN; at day 14,hemorrhage, necrosis and thin fibrotic septa joining central areas of liver were found; at days 21 and 28, thick septa was found; The area-density percentage of collagen fibrosis of the model group was significantly higher compared with the control group at days 7, 14, 21 and 28 (P ＜ 0.05), especially that on the 28th day. There were significant differences in the liver pathologic grading between the two groups at each time point (P ＜ 0.01); the pathologic grading of the model group at day 7 differed from those at days 14 and 28 (P ＜ 0.01). ③The α-SMA positive cells and a transitional hepatic stellate cell were found under the electron microscopy; typical myofibroblast was observed in the model group at day 21 and 28 under the electron microscopy. ④The area-density percentage of collagen fibrosis was positively correlated with the content of serum HA, LN and type Ⅳ collagen (r=0.707, 0.675, 0.662, P＜ 0.01).CONCLUSTON: There are significantly progressional changes in morphological and serum levels of HA, type Ⅳ collagen and LN in different stages of DMN-induced liver fibrosis in rats, moreover, the area-density percentage of collagen fibrosis is positively correlated with the serum levels of HA, LN and type Ⅳ collagen at different stages.